A novel model for endometriosisEndometriosis is an estrogen-dependent, chronic gynecologi-cal...
Transcript of A novel model for endometriosisEndometriosis is an estrogen-dependent, chronic gynecologi-cal...
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炎症・再生 Vol.23 No.1 200396
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Review Article
A novel model for endometriosis
Hirotaka Masuda1,2,*), Tetsuo Maruyama1), Yasunori Yoshimura1),Yumi Matsuzaki2), and Hideyuki Okano2)Departments of 1)Obstetrics and Gynecology and 2)Physiology, Keio University School of Medicine, Tokyo,Japan
Human uterine endometrium has unique properties to regenerate and remodel cyclically throughout the
woman's reproductive life and also gives rise to endometriosis through ectopic implantation of retrograde
shedding including endometrial cells during menstruation. Endometriosis is a common and significant
gynecological disorder which can lead to infertility or a certain type of ovarian cancer. However, the etiology
and pathogenesis still remain uncertain. Previously, we have reported that singly dispersed cells isolated
directly from human endometrium can reconstruct the functional ectopic endometria when transplanted
beneath the kidney capsule of the NOD/SCID/γcnull immunodeficient mouse. In addition to the endometrium-like structure, hormone-dependent changes (e.g. proliferation, differentiation, tissue breakdown and shed-
ding) characteristic of cycling human endometrium can be reproduced in the endometrial reconstruct whose
blood is supplied by human-mouse chimeric vessels. These results indicate that singly dispersed endome-
trial cells have potential applications for tissue reconstitution, angiogenesis, and human-mouse chimeric
vessel formation, providing implications for model mechanisms underlying the establishment of endometriotic
lesions and the physiological endometrial regeneration during the menstrual cycle. Furthermore, the hor-
mone-dependent behavior of the endometrium reconstructed from lentivirally-engineered endometrial cells
expressing a variant luciferase can be assessed noninvasively and quantitatively by in vivo bioluminescenceimaging. Our animal model will provide a powerful tool to investigate the pathophysiology of endometriosis
and also to validate the effect of novel therapeutic agents and gene targeting on endometriosis with the
noninvasive and real-time evaluation system. This animal system can be applied as a unique model for other
various types of neoplastic diseases when the relevant cells are transplanted under the kidney capsule. This
article describes our novel mouse model and in vivo imaging system with overviews of experimental modelsfor endometriosis.
Rec/Acc. 3/9/2010, pp96-102
*Correspondence should be addressed to:Hirotaka Masuda, Departments of Obstetrics and Gynecology and Physiology, Keio University School of Medicine, 35Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. e-mail: [email protected]
Review Article A novel model for endometriosis
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97Inflammation and Regeneration Vol.30 No.2 MARCH 2010
Introduction Endometriosis is an estrogen-dependent, chronic gynecologi-
cal disorder which is characterised by the presence of uterine
endometrial tissue outside of the normal location, mainly on the
pelvic peritoneum. The prevalence of pelvic endometriosis ap-
proaches ~10% in the general female population, and it can cause
severe pain and infertility1). Although it is a benign disease, symp-
toms recur in up to 75% of women within 2 years after surgery
which relieve the pain, and this disorder could increase the risk
of ovarian cancer1,2). However, the etiology and pathogenesis of
endometriosis still remain uncertain. One of the biggest prob-
lems in this area of research is the lack of appropriate in vivomodels for endometoriosis, because rodents that are most com-
monly and conveniently used as animal models do not undergo
menstruation.
In terms of the most widely accepted developmental mecha-
nism for endometriosis, the “implantation theory”3), retrograde
menstruation is essential for peritoneal endometriotic lesions,
even if additional factors are needed for establishment and main-
tenance of endometriosis. In fact, eutopic endometrial cells were
deposited into the peritoneal cavity via retrograde menstruation
and induced the recurrence of endometriosis4). The retrograde
menstruation must have “endometriosis initiating cells” which
can establish and maintain endometriotic lesions, that is to say
the endometriosis initiating cells must originate in human en-
dometrium itself. Because menstrual shedding is a requirement
for the development of endometriosis, the animals except for
some non-human primates don't have spontaneous endometrio-
sis. However, realistically, it is quite difficult to give experimental
evidence on human and non-human primates because of ethical
issues, invasive potential of the experiments and high handling
costs of non-human primates. Therefore, we believe that the in-
vestigation for physiological roles in the pathogenesis of en-
dometriosis is best performed in small animals which have
endometriotic lesions of human origin.
Xenotransplanted mouse models In recent years, immunodeficient mice xenotransplanted with
human endometrial tissue fragments have been used as animal
models of endometriosis5). The survival rate of endometriotic
lesions and the time period for maintenance of well-preserved
endometriotic tissue are limited by the host immune system, as
these factors are shown to improve with increasing immunode-
ficiency potential of the transplanted mice. Human endometrial
tissues have been successfully implanted either subcutaneously
or intraperitoneally into several types of immunodeficient mice
including athymic nude mouse6-11), severe combined immunode-
ficient (SCID) mice12-14), non-obese diabetic (NOD)-SCID mice10)
and NOD/SCID/γcnull (NOG) mice15,16). Additionally, menstrual
endometrium17-19) and endomtriotic tissue20) have also been en-
grafted into nude mice and transgenic recombinase-activating
gene-2 knockout (RAG-2/γ(c)KO) mice respectively.
In terms of its capacity to establish and maintain endometrio-
sis, the endometriosis initiating cell could potentially be a en-
dometrial stem/progenitor cell. More recently, transplantation
of human endometrial epithelial micro-aggregates (organoids),
with dissociated mouse or human endometrial stromal cells, be-
neath the kidney capsule of nude mice developed human en-
dometrial tissue comprising human glands, mouse or human
stroma and myometrium21). While this experiment showed the
importance of epithelial-stromal interactions, it also indicated
the presence of stem/progenitor cells.
Many current models demonstrate sufficient evidence to con-
clude that rare populations of adult epithelial and stromal stem/
progenitor cells exist in human endometrium. However, to study
the pathophysiology of endometriosis or the physiology of hu-
man endometrial stem cells, the models should meet the follow-
ing criteria: 1) the transplanted human graft must be quantita-
tively uniform in each animal, 2) the model should reproduce
both functional and morphological changes characteristic of hu-
man eutopic and/or ectopic endometrium, and 3) it should be
possible to obtain quantitative data of the transplant in real-time,
for a prolonged period, without invasive techniques.
Our mouse model for endometriosis We have recently developed a novel mouse model which fulfil
all these requirements22). When fully dissociated unfractionated
human cells (5x105) were transplanted directly under the kidney
capsule of NOG immunodeficient mice, we observed well-orga-
nized tissues including endometrial and myometrial layers in all
of the transplanted 60 kidneys of ovariectomized NOG mice
which had been hormonally treated with estrogen/progesterone
for 10 weeks (Fig.1). It allows relatively easy production of sev-
eral or even dozens of homogeneous mice from one sample. These
ectopic endometria on the kidneys mimicked endometrial hor-
mone-dependent process such as cellular proliferation, differen-
tiation (decidualization) and tissue breakdown (Fig.1A). Fur-
thermore, the transplants after progesterone withdrawal contained
a large blood-filled cyst like a red spot lesion of endometriosis
(Fig.2) as well as a chocolate cyst generated by subcutaneous
grafting of endometriotic lesions into RAG-2/γ(c)KO mice20).
We have demonstrated that singly dispersed human endometrial
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炎症・再生 Vol.23 No.1 200398 Review Article A novel model for endometriosis
Fig.1 Reconstruction of human endometrium-like tissue from singly dissociated endometrialcells
(A) Macroscopic and microscopic findings of the transplanted site (arrowhead) of NOG mice 10 weeks afterxenotransplantation. H&E staining was performed on the transplanted lesion of NOG mice treated with Estoradiol(E2), Progesterone (P4), or E2+P4 as indicated. The borders between the reconstituted tissue and the mousekidney (K) are indicated by the dotted lines. (B and C) Immunofluorescence staining of the endometrial recon-structs in the E2+P4-treated NOG mice by using antibodies against cytokeratin (Ck) and Vimemtin (Vm) (B), Vmand α -Smooth muscle actine (αSMA) (C), followed by Hoechst (Ho) staining. (Scale bars: 100 μm)Reproduce from ref. 22 with permission. Copyright 2007, National Academy of Sciences, USA.
Fig.2 A large blood-filled cyst like a red spot lesion of endometriosis after progesteronewithdrawal
(A) Macroscopic findings of the transplanted site (arrows). (B) H&E and immunofluorescence staining of thetransplanted lesion. The glandular structure was partially disrupted (arrowheads), and hemorrhage occurred inthe stroma (arrow). A small box marks a region shown at higher magnification in the adjacent panel as indicated.(Scale bars: 100 μm)Reproduce from ref. 22 with permission. Copyright 2007, National Academy of Sciences, USA.
cells can reconstitute ectopic functional endometrial tissues,
mimicking human endometriosis22).
Unique angiogenic potential of humanendometrial cells While host angiogenesis into the human transplants was ob-
served as described previously10,17,19,22-24), our results showed that
human blood vessels invaded into the murine kidney parenchyma
and formed chimeric vessels with the host murine endothelium,
providing a murine blood flow to the transplant (Fig.3). Previ-
ously, it has been reported that human graft vessels disappeared
gradually in other endometriosis models10,19,23,24). Although tu-
mor-derived endothelial cells are known to demonstrate an an-
giogenic ability to grow into immunodeficient mice and formed
vasculatures connected with the host circulation25), this is the first
report of the angiogenesis from human transplants derived from
normal, not cancer, cells into the host tissue22). A lack of NK cell
activity in NOG mice might allow us to see the unique ability of
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99Inflammation and Regeneration Vol.30 No.2 MARCH 2010
Fig.3 Vascularization of the reconstructed en-dometrium
Immunofluorescence staining of the reconstituted endometrialtissue (A) and the mouse kidney parenchyma adjacent to thereconstituted tissue (B and C) by using antibodies againsthuman CD31 (hCD31) and TER-119 (A), hCD31 and Vm (B),or Vm and αSMA (C), followed by Hoechst (Ho) staining. (C)A small box marks a region shown at higher magnification inthe adjacent panel as indicated. (Scale bars: 100 μm)Reproduce from ref. 22 with permission. Copyright 2007, Na-tional Academy of Sciences, USA.
Fig.4 Lentiviral construct for fluorescence and bi-oluminescence and expression of both fluo-rescent and luminescent markers in thelentivirally transduced endometrial cells
(A) Lentiviral construct encoding a dual function CBR luc (aluciferase variant) and Venus (a YFP variant) bicistronic re-porter gene connected via an internal ribosomal entry site(IRES). (B) Phase-contrast and fluorescence microscopy ofthe primary cultures of lentivirally transduced endometrial stro-mal (Upper) and glandular (Lower) cells. (C) A representativeflow cytometric profile of the PI negative fraction (red box atLeft) of the lentivirally transduced endometrial cells consistingof three subpopulations (Right) based on the fluorescentintensity: high (I), low (II), and negative (III) subpopulations.(D) Macroscopic luminescent image of a six-well dish whereeach subpopulation, as sorted in B, was cultured in the corre-sponding well. Non-infected endometrial cells were cultured inthe IV well.Reproduce from ref. 22 with permission. Copyright 2007, Na-tional Academy of Sciences, USA.
Fig.5 Quantitative assessment of the growth of thereconstructed endometria under the kidneycapsules of ovariectomized NOG mice
Representative BLI (top panels) and serial photon count mea-surements (second panel) of NOG mice treated for differentdurations with the various indicated doses of E2 pellets (A),with E2 in combination with daily injections of ICI 182,780, a
pure estrogen antagonist (B), or with cyclic E2+P4 treatment (bottom panel) to induce artificial menstrual cycle-related changes(C). The photon count value of each region of interest (ROI, red circle ) is indicated.Reproduce from ref. 22 with permission. Copyright 2007, National Academy of Sciences, USA.
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炎症・再生 Vol.23 No.1 2003100 Review Article A novel model for endometriosis
endometrial epithelial cells because NK cells play a critical role
in the IL-12-mediated inhibition of angiogenesis26). Importantly,
our observation newly suggests that human endothelial cells/
progenitors in endometrium can migrate, invade, form chimeric
vasculature in host tissue, even that of a different species, and
establish the functional circulatory system. Either way, since the
functional vascularization is absolutely required for maintenance
of endometriotic lesion, the angiogenic potential of the endome-
trial endothelial cells itself may be crucial for establishment and
development endometriosis. Anti-angiogenic therapies have been
recently proposed as a potential alternative treatment for
endometriosis24,27,28). This therapeutic strategy might be further
strengthened by targeting endometrium or endometriosis spe-
cific angiogenesis.
Monitoring system for our model Single-cells transplantation is adequate for several experimental
procedures such as cell selection, genetic engineering, and quan-
titative assessment before transplantation. In our study, we se-
lected the lentivirally infected cells via their flourochrome by
flow cytometry and transplanted a certain number of these in-
fected cells into each kidney (Fig.4). Then, we detected the bi-
oluminescence emitted from living transplanted cells by Biolu-
minescence Imaging (BLI) technique and obtain the quantita-
tive data of the reconstituted tissues in real-time, for a prolonged
period, without invasive techniques (Fig.5). To achieve non-in-
vasive detection and quantification of transplants, dissociated
glandular and stromal cells labelled with lipophylic dye (DiO)29)
and transduced endometrial fragments with GFP cDNA30) were
subcutaneously or intraperitoneally transplanted into nude mice.
However, transplanted cells and tissues scattered or spread un-
expectedly, the emitted lights were diluted with cell division and
the permeability of the light was limited. Therefore, we used the
lentivirus31) capable of introducing and stably expressing the
coding gene and both Venus (a YFP variant)32) and click beetle
red-emitting (CBR) luciferase were encoded on the lentivirus as
reporter genes (Fig.4A). Venus was useful for flow cytometric
selection of lentivirally infected cells (Fig.4B). CBR emitting
light has the capacity to pass through thicker tissues and this
enabled us to detect the viable cells from outside the murine
body by in vivo BLI22). Additionally, kidney capsule transplan-tation allowed a small number of the cells to be localized and
grow three-dimensionally at the relatively superficial location.
Combining our model together with advantages of the Biolumi-
nescence Imaging (BLI) technique33), Firstly, we demonstrated
that the signal intensities reflecting the volume of the recon-
structed tissue were enhanced in an estrogen dose- and time-
dependent manner (Fig.5A). Therefore, the growth behaviour of
the reconstructed tissue was assessed quantitatively and sequen-
tially. Meanwhile, the signal intensity was not increased but rather
decreased 2-3 months after co-treatment with ICI-182,780 (ICI),
a pure estrogen antagonist34) (Fig.5B). These data indicated that
the antagonistic effect of ICI can be noninvasively and success-
fully assessed and this system could be used as a tool for drug
screening. Finally, we could monitor successfully the menstrual
cycle-related dynamic changes of the transplants for an extended
period in a noninvasive, real-time, and quantitative manner
(Fig.5C). Sequential BLI revealed that the signal intensities fluc-
tuated dramatically in accord with the addition and withdrawal
of progesterone (Fig.5C). In particular, tissue breakdown and
regression after progesterone withdrawal and subsequent tissue
regeneration faithfully reflected the decrease and increase in sig-
nal intensity, respectively.
Conclusive Remarks Our animal system showed that singly dissociated human en-
dometrial cells produce the ectopic endometrium. This model
indicated that singly dissociated human endometrial cells had
endometriosis initiating cells and endometrial stem /progenitor
cells. Simultaneously, we proposed the unique animal model is
suitable to study the pathogenesis of endometriosis through
noninvasive, real-time, and quantitative assessment of ectopically
reconstituted endometrium-like tissues. This novel animal model
system can also provide opportunities of drug testing and gene-
target validation in endometriosis22). Furthermore it would be
potentially applicable for other various types of neoplastic dis-
eases when the relevant primary culture cells or cell lines are
transplanted beneath the kidney capsule.
Acknowledgements The author wishes to acknowledge my research group and GayathriRajaraman for their generous assistance.
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